Figure 1.

Methylation-dependent transcriptional silencing of NT5E in breast cancer cell lines. (A) Methylation in the NT5E CpG island correlates with downregulation of mRNA expression. The figure shows MSP analysis of the NT5E CpG island (upper panels) and semi-quantitative RT–PCR analysis of NT5E mRNA and the control gene GAPDH in breast carcinoma cell lines (bottom panels). MSP was performed as described in Materials and Methods. The figure shows unmethylated (U) and methylated (M) reactions for each cell line DNA. Also shown are control U and M DNA samples modified in parallel with the cell line DNA samples. (B) Pyrosequencing analysis of the NT5E CpG island in breast carcinoma cell lines. Pyrosequencing was done as described in Materials and Methods. The level of methylation is represented by the intensity of shading in the circles, each of which represents an individual CpG dinucleotide in the amplified fragment. The mean % CpG methylation in the amplified fragment, together with the methylation-specific PCR analysis is also shown. Abbreviation: ND=not determined.